Internal death programs play significant roles in many diseases. Pathogenic effects can result from inefficient cell death or from inappropriate or excessive death such as that caused by the human immunodeficiency virus (HIV) during AIDS or the SAR-CoV virus during SARS. In this project, we are taking a multifaceted approach to studying molecular mechanisms of both apoptotic and nonapoptotic death programs in lymphocytes as well as other cell types. A major focus of our investigations are death-inducing cell surface receptors in the tumor necrosis factor receptor (TNFR) superfamily such as TNFR1 and CD95/Fas/APO-1. Both receptors play an important role in stimulating both apoptotic and nonapoptotic death of cells principally in immune processes. Little is known about how these alternative death pathways are entrained to receptor signaling. Interestingly, both receptors can have effects beside death such as the induction of transcription factors. We are trying to understand how these receptors stimulate the intracellular machinery that causes cell death in preference to other cellular outcomes. We have discovered that inhibition of caspase-8 in non-lymphoid cells can lead to another form of cell death exhibiting particular cytoplasmic double membrane structures called autophagy. Autophagy is an evolutionarily conserved process from humans to yeast by which cytoplasmic proteins and organelles are catabolized but very little was known about results at the end of autophagy when cells were selecting between autophagic cell death and survival. Mitochondria have a primary physiological role in producing ATP as an energy source, but also regulate cell death. In response to cellular stress, dysfunctional mitochondria produce ROS and other pro-death mediators to initiate programmed cell death pathways, like apoptosis or necroptosis. Mitophagy, a selective form of autophagy, can target dysfunctional mitochondria for lysosomal degradation and protect cells from oxidative damage. This is beneficial for the survival of terminally differentiated cells, like nerve and heart muscle cells. Several regulators of mitophagy, including PINK1, Nix (BNIP3L), and PARKIN have been identified. Mutations or deletions of those genes have been related with a variety of diseases, including ischemia injury in myocardial infarction and stroke, as well as neurodegenerative disease. Hence, understanding the detailed mechanism of mitophagy remains an important goal for improving the diagnosis and treatment of diseases involving mitochondria. Two autosomal recessive Parkinson's disease genes, PINK1 (PTEN induced putative kinase 1) and PARKIN, regulate mitophagic clearance of dysfunctional mitochondria. In healthy cells, PINK1 is constitutively degraded by mitochondrial proteases, such as mitochondria inner membrane protease Presenilin Associated, Rhomboid-Like (PARL) protein. Membrane depolarization of dysfunctional mitochondria inhibits PINK1 degradation, causing it to accumulate and promote mitophagy via recruitment of another familial Parkinson's protein, the E3 ubiquitin ligase PARKIN. However, the detailed mechanism of PINK1 degradation and stabilization remains unclear. We have studied PGAM5, paralog member 5 of a family of highly conserved phosphoglycerate mutases, a 32-kDa mitochondrial protein that apparently lacks phosphotransfer function on phosphoglycerates, but retains activity as a serine/threonine protein phosphatase that regulates the ASK1 kinase. Recently, PGAM5 was found as a downstream mitochondrial target of RIP3 in the necrosis pathway in cancer cells, by recruiting the RIP1-RIP3-MLKL necrosis attack complex to mitochondria. Interestingly, PGAM5 has also been reported as a genetic suppressor of PINK1 in Drosophila, as well as a substrate of PARL. Thus, it is important to establish the in vivo role of PGAM5 involving mitochondria. Using a new strain of knockout mice, we found that PGAM5 is critical for PINK1 stabilization on damaged mitochondria to initiate mitophagy since loss of PGAM5 totally disables PINK1 stabilization. Cells deficient with PGAM5 showed elevated ROS originated from mitochondria, and exacerbated cell necrosis compared to control wild type cells. In stroke and cardiac ischemic-reperfusion injury models, PGAM5-deficient mice showed significantly increased severity of injuries in the brain and heart compared to wild-type mice, indicating that PGAM5 protects against ischemia-reperfusion-induced necrosis. Importantly,in the past year we have found that the PGAM5 KO mice showed a clear movement disorder at one year of age, as well as significant dopaminergic neurodegeneration at an even earlier age. However, the magnitude of DA loss in PGAM5 KO mice is small. In humans and in MPTP-treated mice, a loss of at least 75-80% of the dopamine neurons is required for locomotor problems to be exhibited. The relatively small decrease of DA may due to the compensatory secretion of DA from remaining DA neurons in SN or VTA. Another possibility is that the movement disorder phenotype may also result from dysfunctional DA neurons, a decrease of DA receptor sensitivity or other neurotransmitter systems, which have not yet been identified in PGAM5 KO mice. Thus, mice genetically deficient in PGAM5 exhibit a specific neurological abnormality related with progressive loss of SNpc DA neurons, which mimics a Parkinsonian-like behavioral phenotype. Taken together, our data suggest that PGAM5 promotes PINK1-mediated mitophagy, which could be cytoprotective in ischemic injuries. Our studies have shed new light on the mechanism of PINK1 stabilization by unveiling a new function of the mitochondrial regulatory protein PGAM5. PINK1 stabilization and subsequent parkin recruitment triggers mitophagy, which selectively eliminates dysfunctional mitochondria to protect cells/tissues from oxidative stress and cell death. in our investigations, we have generated new evidence suggesting that mitophagy may contribute to dopaminergic neurodegeneration and movement disorders in experimental animals by the role of PGAM5 in these processes. Contrary to a previous study in drosophila, in a mammalian system, we found PGAM5 protects dopamine neurons from degeneration, presumably by promoting PINK1 stabilization. Consistent with a mitochondrial pathogenesis for Parkinsons disease, PINK1 deficiency in drosophila causes energy depletion, shortened lifespan, and dopamine neuron degeneration. Aged PINK1 deficient mice show impaired neural activity similar to PGAM5 KO mice, but without DA neurodegeneration; and mutations in PINK1 and parkin predispose to the movement disorders and DA neurodegeneration that characterizes familial Parkinsons disease in humans. The new role for the mitochondrial protein PGAM5 in dopamine neuron pathology, lends further weight to the mitochondrial theory of Parkinson's pathogenesis that has been developed by Dr. Richard Youle in NINDS.